Measurement of antimicrobial activity under reducing conditions in a modified radial diffusion assay

protocol.md

Authors: Bjoern O. Schroeder & Jan Wehkamp

Abstract

This protocol describes the measurement of antimicrobial activity under reducing conditions against anaerobic bacteria of the human normal flora. The protocol is modified from the original description by Robert Lehrer et al. (1991), which is well established for the analysis of antimicrobial peptides with aerobic bacteria. To perform analysis for facultative and strict anaerobes we modified the assay by the addition of the reducing agent DTT to assay medium and performed incubation steps in an anaerobic jar. It was successfully used with analysis of Bifidobacteria, Lactobacilli, Bacteroides and Escherichia coli and might be adapted to other anaerobic bacteria. Several antimicrobial peptides have been tested with that system: while most of them showed less antimicrobial activity under reducing conditions, human beta defensin-1 was only active under these conditions against some of the mentioned strains.

Introduction

This protocol describes the measurement of antimicrobial activity under reducing conditions against anaerobic bacteria of the human normal flora. The protocol is modified from the original description by Robert Lehrer et al. (1), which is well established for the analysis of antimicrobial peptides with aerobic bacteria. To perform analysis for facultative and strict anaerobes we modified the assay by the addition of the reducing agent DTT to assay medium and performed incubation steps in an anaerobic jar. It was successfully used with analysis of Bifidobacteria, Lactobacilli, Bacteroides and Escherichia coli (2) and might be adapted to other anaerobic bacteria. Several antimicrobial peptides have been tested with that system: while most of them showed less antimicrobial activity under reducing conditions, human beta defensin-1 was only active under these conditions against some of the mentioned strains.

Reagents

1. Type I low EEO-agarose (Sigma-Aldrich, #A6013)

• Tryptic Soy Broth (TSB) powder (Difco, #211825)
• monobasic sodium phosphate buffer NaH2PO4 ∙ H2O (MW = 137.99)
• dibasic sodium phosphate buffer Na2HPO4 ∙ 2 H2O (MW = 177.99)
• Dithiothreitol (DTT) 1 M stock solution in H2O from powder (Carl Roth, #6908.1)
• HCl (1:10 and 1:100 dilutions in H2O)
• Resazurine (Sigma-Aldrich, #R7017) stock solution 1 mg/ ml in H2O
• Bacteria to analyze
• Peptides to analyze (dissolved in H2O or 0.01% acetic acid)
• Columbia Blood Agar plates (or similar growth medium)

Equipment

1. three 100 ml bottles

• five 500 ml bottles
• 50 ml Falcon tubes
• square-dish 100×100 x 15 mm (BD Falcon, # 351112)
• sterile glass pipettes 1 ml, 5 ml, 10 ml
• gel puncher (for instance biopsy punch (Stiefel))
• 37°C anaerobic incubator or system (Oxoid #AG0025, #AN0025, #BR0055)
• centrifuge for 50 ml Falcon tubes
• autoclave
• microwave
• water-bath maintained at 46°C
• spectral-photometer at 620 nm

Procedure

A. Prepare media and reagents

1. 1x Tryptic Soy Broth (TSB):

• Dissolve 15 g of TSB powder in 500 ml ddH2O, autoclave
• Phosphate buffer 100 mM, pH 7.4
  • Prepare 200 mM monobasic phosphate buffer (NaH2PO4 ∙ H2O) (MW = 137.99): dissolve 13.8 g of NaH2PO4 in 500 ml ddH2O
  • Prepare 200 mM dibasic phosphate buffer (Na2HPO4 ∙ 2 H2O) (MW = 177.99): dissolve 17.8 g of Na2HPO4 in 500 ml ddH2O
  • Mix 47,5 ml of monobasic NaP with 202,5 ml dibasic NaP, add 250 ml H2O, autoclave
• Phosphate buffer 10 mM, pH 7.4
  • Dilute 100 mM Phosphatpuffers 1:10 with H2O, final volume of 100 ml, autoclave
• Underlay “low nutrition” gel (5 gels)
  • add 0.5 g EEO-agarose into 100 ml bottle
  • add 0.5 ml 1x TSB
  • add 5 ml of Phosphate buffer 100 mM, pH 7.4
  • add ddH2O to a final volume of 50 ml (Note: If melting of the gel in the microwave causes significant loss of liquid it is possible to add more ddH2O in order to keep a remainder of 50 ml after microwaving.)
  • adjust pH to 7.4 with diluted HCl (1:100 or 1:10) (Note: depending on the number of gels you prepare only few drops might be required.)
  • autoclave in 100 ml bottle for 20 min at 121°C, store for short term at room temperature
• Overlay „high nutrition“ gel (5 gels)
  • add 0.5 g EEO-agarose into 100 ml bottle
  • add 3 g TSB powder
  • add 5 ml of Phosphate buffer 100 mM, pH 7.4
  • add ddH2O to a final volume of 50 ml (Note: If melting of the gel in the microwave causes significant loss of liquid it is possible to add more ddH2O in order to keep a remainder of 50 ml after microwaving.)
  • autoclave in 100 ml bottle for 20 min at 121°C, store for short term at room temperature

B. Bacterial Preparation

1. Prepare anaerobic bacteria from kryo-culture

• Incubate on Columbia Blood-Agar plates in an anaerobic jar for about 48 hours
• Transfer bacteria into 50 ml falcon containing 10 ml 1x TSB
• Place Falcon without lid into anaerobic jar, incubate for about 16 hours at 37°C without shaking. (Note: Since different bacterial strains behave differently optimal culture conditions should be optimized. Some bacteria might require shorter incubation times while others might require more time to produce optimal numbers of cells required for the assay.)

C. Modified Radial Diffusion Assay

1. Pre-chill centrifuge at 4°C

• Place sterile phosphate buffer 10 mM, pH 7.4 on ice
• Melt Underlay gel in microwave (Note: Mix gently to produce homogenous preparation.)
• Transfer to 50 ml Falcons with 10-ml aliquots each; maintain gel liquid by placing in 46°C water bath
• 5 – 20 µl DTT can now be added into liquid gel from the 1 M stock solution. (Note: It is important to use freshly prepared DTT since repeated freezing and thawing impairs its function. Furthermore, some bacterial strains tolerate higher amounts of DTT while others do not. We obtained best results between concentrations of 0.5 and 2 mM (final) whereas 10 mM were mostly lethal. Depending on bacteria used different amounts of DTT might lead to best results.
• 10 – 20 µl resazurine can be added into liquid gel (final concentration 1 – 2 µg/ ml). (Note: The addition of this redox-indicator is a good opportunity to control reducing conditions in the underlay-gel. A “classical gel” should stain light blue while a reduced gel should look pink.)
• Centrifuge bacterial overnight-culture in Falcons for 10 minutes at 4°C and 900 x g
• Wash bacterial sediment in 10 ml ice-cold sterile phosphate buffer 10 mM, pH 7.4
• Centrifuge bacterial culture for 10 minutes at 4°C and 900 x g
• Resuspend sediment in 5 ml ice-cold sterile phosphate buffer 10 mM, pH 7.4
• Determine Optical Density at 620 nm against a buffer control (10 mM phosphate buffer)
• Adjust to OD620nm = 0.1 and use 150 – 300 µl for assay (Note: The optimal volume of bacterial culture should be determined after the first experiments depending on the bacterial lawn obtained after the over-night incubation of the assay plates. In our hands it was optimal to use 150 µl for Bifidobacteria and 300 µl for Lactobacilli. For fast-growing bacteria like E. coli the user should refer to the classical protocol of Lehrer et al. (1))
• Add bacterial suspension into 10 ml of warm, prepared underlay gel, mix gently. (Note: It is important that the gel is not too hot, which would have an effect on or kill bacterial cells, and not too cold since the gel would solidify. A temperature range between 42°C and 46°C should be fine for mixing with bacterial cells.)
• Quickly pour the gel containing bacteria into square-petri-dishes, placed on an even surface, and let it solidify for about 30 minutes at room temperature. (Note: By letting a small opening between plate and its lid moisture level may be reduced, which improves punching of the wells later on. For strict anaerobic bacteria solidification time should be minimized).
• Punch 6×6 wells into the gel and discard gel pieces with a sterile pipette tip or Pasteur-pipette connected to a vacuum pump
• Fill up to 5 µl of proteins, dissolved in H2O or 0.01% acetic acid, into the wells. Include negative control (H2O or 0.01% acetic acid) and positive control (for example lysozyme, depending on bacteria tested)
• Let protein solutions diffuse into the gel (short storage at 37°C or room temperature) and place plates into anaerobic jar for 3 hours.
• Melt overlay gel in microwave, transfer to 50 ml falcons, 10 ml each, maintain at 46°C
• Add DTT and resazurine, according to corresponding underlay gel
• Pour 10 ml of nutrient-rich overlay gel onto each underlay gel and let solidify
• Place plate upside-down in anaerobic incubator until bacterial lawn is clearly visible (16 to 48 hours)

Troubleshooting

1. No bacterial lawn after incubation.

– Bacteria added to liquid gel might not have been viable. Plate some remaining microliters of the over-night culture on Columbia Blood Agar plates and incubate anaerobically as a control – Liquid gel might have been too hot. Reduce maintaining temperature or wait a little bit longer before mixing with bacteria – For strict anaerobic bacteria oxygen exposure might have been to long – DTT concentration might have been too high

2. Bacterial lawn is too thick, no inhibition zones are visible

– Use less volume of bacterial culture for underlay gel – Reduce incubation time for the primary overnight culture

3. No inhibition zones are visible

– Inappropriate positive control (for example lysozyme does not work for Bacteroides vulgatus) – Proteins/ Peptides are not functional under reducing conditions – Too much moisture in the underlay gel has pressed protein solution out of the wells

References

1. Lehrer, R.I., et al. Ultrasensitive assays for endogenous antimicrobial polypeptides. J. Immunol. Methods 137 (2), 167-173 (1991)

• Schroeder, B.O. et al. Reduction of disulfide bonds unmasks potent antimicrobial activity of human β-defensin 1. Nature, DOI: 10.1038/nature09674

Associated Publications

Reduction of disulphide bonds unmasks potent antimicrobial activity of human β-defensin 1. Bjoern O. Schroeder, Zhihong Wu, Sabine Nuding, Sandra Groscurth, Moritz Marcinowski, Julia Beisner, Johannes Buchner, Martin Schaller, Eduard F. Stange, and Jan Wehkamp. Nature 469 (7330) 419 - 423 20/01/2011 doi:10.1038/nature09674

Author information

Bjoern O. Schroeder & Jan Wehkamp, Wehkamp Lab, IKP Stuttgart

Competing financial interests: The authors filed a patent application on the therapeutic use of reduced human beta-defensin 1 (hBD-1).

Correspondence to: Jan Wehkamp (jan.wehkamp@ikp-stuttgart.de)

Source: Protocol Exchange (2011) doi:10.1038/protex.2010.204. Originally published online 20 January 2011.